Biology subjects

Shaltz, S.

Publications and source records attributed to Shaltz, S..

4 recordsLinked to original sources

bioRxiv2025

Temperature and genetic background drive mobilization of diverse transposable elements in a critical human fungal pathogen

Transposable elements (TEs) are key agents of genome evolution across all domains of life. These mobile genetic elements can cause mutations through transposition or by promoting structural rearrangements. Stress conditions can amplify TE mobility, either by impairing TE suppression mechanisms or through stress-induced interactions between transcription factors and TE sequences, offering a route for rapid genetic change. As such, TEs represent an important source of adaptability within populations. To investigate the interplay between environmental stress and eukaryotic TE dynamics relevant to infectious disease, we examined how heat stress and host-mimicking medium (RPMI) affect TE mobility in the global human fungal pathogen Cryptococcus neoformans, using a collection of clinical and environmental isolates. Using a selection-based screen, we captured the mobilization of seven distinct mobile element families, encompassing diverse retrotransposons and DNA transposons, whose insertions conferred antifungal resistance. This includes a novel element, CNEST, which belongs to the CACTA, Mirage, Chapaev (CMC) supergroup. Heat stress at human body temperature (37{degrees}C) significantly increased the mobilization of a subset of these TEs, leading to higher rates of acquired antifungal resistance. Whole-genome assemblies revealed that, compared to retrotransposons, DNA transposons were hypomethylated and approximately uniformly distributed throughout the genome, features that may contribute to their frequent mobilization. We further assessed TE-driven genomic changes within hosts using serial isolates from patients with recurrent cryptococcal infections and from isolates passaged through mice. While we observed evidence of TE copy number changes near chromosome ends, we found no indication of TE-mediated alterations near gene-coding regions across any of the serial isolates. Finally, TE mobility was isolate- and strain-dependent, with significant variation even among clonally related strains collected from the same patient, emphasizing the role of genetic background in shaping TE activity. Together, these findings reveal a complex and dynamic relationship between environmental stress, genetic background, TE type-specific epigenetic regulation, and TE mobility, with important implications for adaptation and acquired antifungal resistance in C. neoformans. AUTHOR SUMMARYTransposable elements (TEs) are mobile segments of DNA that can "jump" from one place to another in a genome, causing genetic changes that can impact fitness and evolution. In this study, we investigate how different environmental conditions influence TE mobility in the pathogenic yeast Cryptococcus neoformans, which is globally distributed in the environment and causes life-threatening infections. We compared genetically diverse strains and found that growth at higher temperatures, specifically human body temperature (37{degrees}C), increases the mobility of some TEs. In contrast, other TEs were more active at lower temperatures (30{degrees}C), indicating distinct pathways of temperature regulation for different TE groups. By examining C. neoformans isolates from patients with recurrent infections, we observed genetic changes by one TE group that likely occurred during infection. We also found that TE groups are distributed differently across the genome and show varying levels of DNA methylation, a modification that can suppress their activity and control TE mobility in the cell. Overall, this work shows that environmental stresses, such as increased temperature, can accelerate genetic change by promoting TE mobilization. These changes may contribute to how C. neoformans adapts during infection or in the environment.

genetics↗

bioRxiv2023

Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast

A targeted double-strand break introduced into the genome of Saccharomyces cerevisiae is repaired by the relatively error-prone nonhomologous-end joining (NHEJ) pathway when homologous recombination is not an option. A ZFN cleavage site was inserted out-of-frame into the LYS2 locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5' overhangs. Repair events that destroyed the cleavage site were identified either as Lys+ colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+ events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol {sigma} and Pol 11. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required TLS polymerases as well as the exonuclease activity of the replicative Pol DNA polymerase. Survivors were equally split between NHEJ events and 1 kb or 11 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3' tails. Finally, NHEJ was more efficient in non-growing than in growing cells and was most efficient in G0 cells. These studies provide novel insight into the flexibility and complexity of error-prone DSB repair in yeast.

molecular biology↗

bioRxiv2020

Recombinational repair of nuclease-generated mitotic double-strand breaks with different end structures in yeast

Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3 overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol {delta}-mediated removal of a mismatch 8 nucleotides from a 3 end affects recombination outcomes and whether the presence of a 3 versus 5 overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers versus noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3 end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3 versus 5 overhangs differs, we compared the well-studied 3 overhang created by I-SceI to a 5 overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN-than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

genetics↗

bioRxiv2020

Trapped Topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5 end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2- F1025Y,R1128G) whose product generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2- F1025Y,R1128G allele is associated with a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that over-express the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2. SIGNIFICANCE STATEMENTDNA-strand separation during transcription and replication creates topological problems that are resolved by topoisomerases. These enzymes nick DNA strands to allow strand passage and then reseal the broken DNA to restore its integrity. Topoisomerase II (Top2) nicks complementary DNA strands to create double-strand break (DSBs) intermediates that can be stabilized by chemotherapeutic drugs and are toxic if not repaired. We identified a mutant form of yeast Top2 that forms stabilized cleavage intermediates in the absence of drugs. Over- expression of the mutant Top2 was associated with a unique mutation signature in which small (1-4 bp), unique segments of DNA were duplicated. These de novo duplications required the nonhomologous end-joining pathway of DSB repair, and their Top2-dependence has clinical and evolutionary implications.

molecular biology↗